Bacteria have evolved complex pathways for communicating and competing with each other. Such mechanisms ultimately dictate the composition of microbial communities important in a wealth of environmentally and medically relevant contexts. The type VI secretion system (T6SS) is an emerging model system for the study of bacterial interactions. We recently demonstrated that the T6SS facilitates delivery of protein effectors to other Gram-negative bacteria. The system is found in hundreds of sequenced proteobacterial genomes and its activity can have dramatic consequence on target cell populations. Though the T6SS is often described as a bacteriophage-like pathway that punctures recipient cells and injects toxins, a functioning T6S apparatus and the effects it exerts on recipient cells have yet to be directly observed. This proposed collaborative project seeks to use quantitative time-lapse fluorescence microscopy to directly visualize and quantitatively characterize the action and potency of T6S-based bacterial intoxication with single- cell resolution. In the first aim, we will utilize single cell analyses t identify and quantitatively describe the effects of T6S on recipient bacteria. Our methodology will allow us to determine the spectrum of targeting consequences and measure the contact-dependent killing rate, which is a key determinant of cell fitness, cannot be directly measured in traditional plate of liquid competition assays. The second aim of the proposal is to establish the cellular targeting mechanism of the T6S apparatus by defining a causal relationship between target cell intoxication and the localization and dynamic behavior of defined T6S components. By studying classes of proteins associated with T6S substructures, we will decipher the targeting mechanism of the T6S apparatus (random versus directed) and identify events diagnostic of effector delivery. Critically, the research groups supported by this proposal bring together the interdisciplinary and complementary expertise needed to successfully complete the work. The proposal includes strong preliminary data demonstrating feasibility and proof-of-concept of an innovative strategy for the quantitation of bacterial interactions with single-cell resolution. We expect this work to both generate important insights into bacterial secretion as well as develop an analytical framework that will be generally applicable to the quantitative analysis of bacterial cellular interactions.